This invention relates to a new method for determining catecholic compounds and their related compounds and an apparatus for the determination thereof by this method.
Catecholic compounds are found in the animal and vegetable kingdoms, and among them, catecholamines, which play an important role among mammals, may be cited as typical. It is of utmost importance, therefore, in the fields of medicine, pharmacy, biochemistry, pharmacology, neurochemistry, etc. to determine the fluctuation in a biological substance in the amount of catecholamines (such as dopamine, norepinephrine or epinephrin) and their related compounds or their metabolites (3-methoxytyramine, normetanephrine, metanephrine, homovanillic acid, vanillylmandelic acid, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylglycol, etc.).
While catecholamines and their metabolites are present in very small quantities, various methods for the determination of catecholamines have recently been put into practical use. The methods applied are selected according to their effectiveness for the intended purpose and include, for example, methods using such techniques as gas chromatography (Chem. Pharm. Bull., 16:699-701, 1968), gas chromatography-mass spectrometry (Sci, 176: 177-180, 1972), high-performance liquid chromatography (Life Sci., 14:311-322, 1974) and the enzymeisotope method (Circ. Res., 26:53-57, 1970).
There has been little study, however, on the determination of the metabolites, and no suitable method for accurate and highly sensitive determination thereof has yet been established. Recent advancements in high-performance liquid chromatography have made it possible to separate the components of a substance which may contain the metabolites. Thus, as described in "J. Chromatogr., 116:240-243, 1976," the analysis of such a substance may be carried out using the most recent advancement in high-performance liquid chromatography in combination with spectrophotometry. However, when absorbance at 280 nm is used as described in the above-cited paper, a problem has been encountered in respect of the selectivity for quantifying the metabolites from biological samples composed of many components including mono-phenolic compounds which have maximum absorption around 280 nm. In addition, the described method is disadvantageous in that the sensitivity also is insufficient for quantifying extremely small amounts of the metabolites as in normal human urine.
It is described in "Nature, MacMillan Journals Limited (170:247-250, 1952)" that the solution of mixed ferric chloride and potassium ferricyanide was used as a color developer in paper chromatographic analysis of phenols. The reference only states that with such compounds the instaneous development of a color will be produced (refer to the 14th line from the bottom, left column, page 250). Thus, it has not been known that the reactivities therewith are different depending upon the types of phenols.